The protein tyrosine kinase, Lck plays a pivotal role in mediating signals generated through the T cell antigen receptor complex (TCR). Lck is a member of the Src family of tyrosine kinases. Several structural features of Lck mediate its function, including its catalytic domain, its src- homology 2 (SH2) and 3 (SH3) domains and its covalent modification by palmitic acid. Much recent interest in palmitoylation as a regulatory modification of Src kinases has been generated and a significant portion of this revised application is dedicated to investigating the role of palmitoylation in Lck function. Strategies are presented for determining the effect of surface receptor (e.g., TCR and CD4) engagement on the palmitoylation rate of Lck and for investigating the contribution hydrophobicity makes to Lck function by utilizing a novel synthetic analog of palmitic acid. Lck is also modified by phosphorylation on both serine and tyrosine residues and its tyrosine phosphorylation regulates its catalytic activity. In contrast little information is available concerning the role serine phosphorylation plays in mediating Lck function. Lck becomes serine phosphorylated following TCR ligation and at the mitotic phase of the cell cycle. We propose to investigate the role serine phosphorylation plays in Lck functioning by generating mutants of Lck that lack serine phosphorylation sites as well as mutants that mimic serine phosphorylated Lck. Strategies are presented: 1) to identify the sites of mitotic serine phosphorylation in Lck and the responsible kinase(s); 2) to define a role for the serine phosphorylation of Lck; and, 3) to define and characterize functions for mitotic Lck. These studies will further our understanding of the regulatory role of palmitoylation and serine phosphorylation in Lck function as well as our understanding of the function of Lck at mitosis. They will contribute information to what appears to be an emerging linkage between signaling pathways initiation entry into the cell cycle and events regulating the final stages of cell division.